PROJECT SUMMARY This application proposes to develop fundamental genomic and bioinformatic resources that will better enable studies of a model amphibian, the Mexican axolotl (Ambystoma mexicanum). The axolotl is used in several areas of biomedical research including regenerative medicine and stem cell biology. Axolotls show a remarkable ability to regenerate damaged body parts, including whole limbs, spinal cord, and brain. Understanding how axolotls regenerate complex tissues may reveal mechanisms to achieve endogenous regeneration in humans. Specific Aim 1 will continue efforts to sequence the large axolotl genome, which is 10x larger than the human genome. An innovative chromatin capture approach (Hi-C) will be used to develop DNA libraries that contain information for scaffolding unordered genomic fragments. Accomplishment of this aim will yield a highly contiguous and annotated genome assembly, an essential resource for any model organism. Specific Aim 2 will perform the first epigenetic studies of the axolotl genome. These studies will characterize changes in histone modifications, whole genome methylation patterns, and local chromatin interactions during limb regeneration, leveraging an existing transcriptional dataset that provides robust temporal resolution of gene expression. These studies will identify chromatin modifications and interactions that arise or are remodeled during critical phases of limb regeneration. Specific Aim 3 will develop genomic sequence for a second salamander model, the red-spotted newt (Notophthalamus viridescens). The newt is a logical choice for sequencing because it too serves as a model organism in tissue regeneration research, and its phylogenetic distance from axolotl is ideal for identifying conserved and lineage-specific non-coding sequences that may function to regulate transcription during regeneration. Thus, the proposed newt genomic sequence will broaden the salamander toolkit, increasing the likelihood for future clinical applications in biomedicine. Broad impact will be achieved by sharing genomic and epigenetic data from all three Specific Aims through an existing public website (Sal-Site). Overall, the project will greatly enhance salamander models for biomedical research.